Project description:We have evidence to demonstrate that hPSCs enter mesoderm or endoderm lineages dependent on the dose of WNT signaling activation. To understand the underlying mechanism of CHIR99021-induced definitive endoderm (DE) differentiation, we collected hPSCs treated by CHIR99021 for 21 hours and performed RNA sequencing, to investigate the dynamics of transcriptomes in every three hours post CH treatment.
Project description:Optogenetics is a rapidly advancing technology that combines photochemical, optical and synthetic biology techniques to control cellular behaviour and physiology. Combining sensitive light responsive optogenetic intervention tools with human pluripotent stem cell differentiation models has the potential to refine differentiation and unpick the processes by which morphogenetic signals direct tissue patterning, organisation and cell specification. Here, we utilised an optogenetic bone morphogenetic protein (BMP) signalling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We initially engineered light-sensitive hESCs through CRISPR/Cas9-mediated integration of the optoBMP system into the AAVS1 locus. Activation of optoBMP with blue light in lieu of BMP family growth factors during differentiation resulted in activation of BMP signalling pathway mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells left in the dark. Furthermore, cells differentiated with light were capable of forming chondrogenic pellets consisting of a hyaline-like cartilaginous matrix.
Project description:Smad1/5 are transcription factors that engage in BMP-induced transcription. We determined and analyzed Smad1/5 binding sites by ChIP-sequencing. We used expression microarrays to compare the Smad1/5 binding sites identified by ChIP-seq to BMP-induced gene expressions. HUVECs were treated with BMP for RNA extraction and hybridization on Affymetrix microarrays.
Project description:We report a bulk RNA sequencing analysis of a population containing different mesendoderm progenitor cells derived from hiPSCs in 3D environment. Specifically, 3D aggregates of hPSCs were differentiated into cardiac progenitor cells through WNT signaling activation from day 0 to day 1 and WNT signaling inhibition from day 3 to day 5 of differentiation, which allowed further efficient and reproducible specification into hiPSC-derived CMs. This represents the control protocol from which samples from D3 (D3N) and D5 (D5N) were collected. Additionally, adaptation of this control protocol by performing WNT and BMP signalling pathways activation from day 3 to day 5 of differentiation, allowed the modulation of the progenitor cells population to a more posterior splanchnic mesoderm which favours further specification of those progenitors into hiPSC-derived pro-epicardium/septum transversum cells. This represents the CB protocol from where samples from D3 (D3CB) and D5 (D5CB) were collected.
Project description:Melanoma is a highly aggressive skin cancer, which has the fastest and second fastest growing incidence of any cancer in men and women, respectively. Although melanoma accounts for 5% of skin cancers, it is responsible for 80% of deaths related to skin cancers. The lifetime risk of melanoma is currently estimated to be 1 in 75 and is rising every year. The present study shows that inhibition of bone morphogenetic protein (BMP) signaling pathway is a potential target for treating metastatic melanoma. To characterize the underlying signaling mechanism, comparative proteomic profile of the mouse melanoma tissue treated with and BMP inhibitor (LDN193189) with untreated controls was performed using liquid chromatography-tandem mass spectrometry. Our mass spectrometry data analysis leads to identification of a total of 3231 nonredundant proteins. Of the 3231 proteins, 117 were up-regulated (fold change≥ 1.5) while 14 were down-regulated (fold change ≤ 0.66). Pathway enrichment analysis showed that altered proteins were primarily grouped into: regulation of actin cytoskeleton, phagosomes, proteoglycans in cancer, Focal adhesion, ECM receptor interaction and fatty acid metabolism pathway. Our studies suggest that inhibition of the BMP signaling cascade with small molecule inhibitors decreased the expression of the MT1 and MT2 (metallothionine 1 and 2) which are well known for the antiapoptotic, antioxidant, proliferative, and angiogenic effects in cancer.